In the decade and a half since the discovery of the underlying mutations in the keratin 5 (K5) and K14 genes in the skin disorder epidermolysis bullosa simplex (EBS), there has been a plethora of additional research identifying the genetic bases of many epithelial fragility disorders with 20 of the 54 human keratin genes now linked to genetic diseases, including the skin and nail disorder pachyonychia congenita (PC). Despite these significant discoveries, little progress has been made in developing therapeutics for these genodermatoses. We have recently discovered that the inducible keratins (K6a, K6b, K16, and K17), mutation in any of which can result in PC, contain regulatory motifs in their 5' untranslated regions that may make them susceptible to downregulation following treatment with the macrolide rapamycin (sirulimus). Indeed, treatment of human HaCaT keratinocytes with rapamycin led to a 90% decrease in K6a expression as determined by western blot analysis. A small rapamycin off-label trial in three PC patients showed marked reduction in PC symptoms when therapeutic rapamycin blood trough levels were achieved, with striking resolution observed for painful neurovascular structures on the soles of the feet. These patients, unfortunately, were affected by the well-known side effects arising from systemic administration of rapamycin and two patients withdrew prematurely from the study for these reasons. The purpose of this proposal is to define the pathway involved in downregulation of keratin gene expression following rapamycin treatment in keratinocytes and to develop rapamycin prodrug conjugates (including skin penetrating rapamycin derivatives) that can be applied topically at high local concentrations, thereby avoiding the undesirable side effects resulting from systemic treatment. The wealth of experience achieved by Dr. Paul Wender and colleagues at Stanford University (and Cellgate) in successfully converting cyclosporine, an agent that does not penetrate skin, into cyclosporine transporter conjugates that readily penetrate multiple layers of human skin using oligoarginine or lipid transporters with linkers that release free drug will be incorporated into the rapamycin-conjugate design. The ability to topically deliver rapamycin derivatives to downregulate expression of the inducible keratins may have much broader application, as rapamycin has been shown to be of benefit to patients suffering from psoriasis. PUBLIC HEALTH RELEVANCE Despite the exciting discoveries of the underlying genes and mutations responsible for a large number of genodermatoses, few if any novel clinical treatments have emerged. The purpose of this proposal is to exploit the recent discovery that rapamycin selectively inhibits expression of keratins involved in the skin disorder pachyonychia congenita. In Phase 1, we validate the rapamycin-sensitive pathway involved in keratin mRNA translation and prepare rapamycin prodrugs with skin-penetrating transporter elements and in Phase 2, we utilize animal models to optimize formulations to topically delivery rapamycin and rapamycin conjugates in preparation for a clinical trial. In theory, the platform technology developed should be applicable to other skin disorders including psoriasis caused by over- or inappropriate expression of rapamycin-sensitive keratins. [unreadable] [unreadable] [unreadable]